The ultimate goal of our research is to understand the balance between immune stimulation and immune evasion of cytomegalovirus (CMV) and to use this understanding for the development of new treatments and vaccines. One of the major challenges for CMV vaccine development is the fact that CMV establishes secondary persistent infections in CMV-immune individuals despite the presence of significant antibody and T cell responses. However, this unique ability also represents an opportunity for the design of CMV-based vaccine vectors that can be used repeatedly despite pre-existing immunity to the vector. We recently demonstrated that super-infection by CMV is enabled by the viral US2-11 glycoproteins -US2, US3, US6 and US11- all of which inhibiting antigen presentation by major histocompatibility complex class I (MHC-I) to CD8+ T cells. These observations suggest that CMV lacking the US2-11 region can be used to monitor whether a CMV-specific CD8+ T cell response is "protective", i.e. able to control primary viremia as observed for sero- positive individuals. A goal of this proposal is therefore to define the CD8+ T cell response required for protection against US2-11-deleted virus and to correlate these results with protection against primary infection with wildtype virus. A further goal is to determine the contribution of each individual immunevasin encoded in the US2-11 region in promoting super-infection. These goals will be achieved in three specific aims: In aim 1 we will determine whether a single-cycle virus can induce a T cell effector memory response that protects against super-infection with US2-11 deleted virus and limits viremia upon challenge with wildtype virus. In aim 2, we will determine whether CD8+ T cell responses directed against a single open reading frame of CMV are sufficient to prevent super-infection by CMV lacking US2-11. In aim 3, we will determine whether CMV with iteratively smaller deletions in the US2-11 region is able to overcome pre-existing immunity. This work will challenge existing paradigms, specifically the assumptions that live CMV vaccines need to be based on replicating CMV and that single subunit vaccines confer protective T cell responses. We further anticipate to develop new and improved ways to test CMV vaccines and to improve CMV-based vectors. Therefore, we believe that our research will have a significant and lasting impact on the development of CMV vaccines and CMV-based vaccine vectors. PUBLIC HEALTH RELEVANCE: Human cytomegalovirus (HCMV) is the most frequent infectious cause of birth defects and the development of a CMV vaccine has been given high priority by the Institute of Medicine and National Vaccine Advisory Committee. We discovered immune evasion mechanisms that enable CMV to overcome pre-existing immunity which explains why it has been so difficult to develop a vaccine that protects against infection. In this proposal we will evaluate the more realistic goal is to develop vaccines that protect against the symptoms of primary infection by using attenuated viruses or subunit vaccines.